Biophysics

Proper genome organization is essential for genome function and stability. Disruptions to this organization can lead to detrimental effects and the transformation of cells into diseased states. Individual chromosomes and their subregions can move or rearrange during transcriptional activation, in response to DNA damage, and during terminal differentiation. Techniques such as fluorescence in situ hybridization (FISH) and chromosome conformation capture (e.g., 3C and Hi-C) have provided valuable insights into genome architecture. However, these techniques require cell fixation, limiting studies of the temporal evolution of chromatin organization in detail. Our understanding of the heterogeneity and dynamics of chromatin organization at the single-cell level is still emerging. To address this, clustered regularly interspaced short palindromic repeats (CRISPR)/dead Cas9 (dCas9) systems have been repurposed for precise live-cell imaging of genome dynamics. This protocol uses a system called CRISPRainbow, a powerful tool that allows simultaneous targeting of up to seven genomic loci and tracks their locations over time using spectrally distinct fluorescent markers to study real-time chromatin organization. Multiple single-guide RNA (sgRNA), carrying specific RNA aptamers for labeling, can be cloned into a single vector to improve transfection efficiency in human cells. The precise targeting of CRISPRainbow offers distinct advantages over previous techniques while also complementing them by validating findings in live cells.

Determining the oligomeric state of membrane proteins is critical for understanding their function. However, traditional ex situ methods like clear native gel electrophoresis can disrupt protein subunit interactions during sample preparation. In situ methods such as stepwise photobleaching have limitations due to high expression levels and limitations of optical resolution in microscopy. Super-resolution microscopy techniques such as single-molecule localization microscopy (SMLM) have the potential to overcome these limitations, but the stochastic nature of signals can lead to miscounting due to over-expression, background noise, and temporal separation of signals. Additionally, this technique has limited application due to the limited selection of fluorescent labels and the demanding control of laser power. To address these issues, we developed a dual color colocalization (DCC) strategy that offers higher tolerance to background noise and simplifies data acquisition and processing for high-throughput and reliable counting. The DCC strategy was used to determine the oligomeric states of membrane proteins of the SLC17 and SLC26 family with SMLM, providing a robust and efficient method for studying protein interactions.

Graphical overview

(A) Illustration of the principle for determining the oligomeric state of protein complexes with dual color colocalization–single-molecule localization microscopy (DCC-SMLM). In the inset, as an example, a dimeric protein (brown) is labeled with a marker (M) and an indicator fluorescent protein (F) on each of its two subunits. The overall probability of detecting the dimer with SMLM, as denoted by R, the colocalization ratio, is equal to the ratio of the number of colocalized marker and indicator clusters (NMF) to that of the marker clusters (NM). The plot shows the linear relationship of the oligomeric state (n) vs. the natural logarithm of 1 subtracted by the colocalization ratio, supplemented by the equation of the fitting curve, in which p denotes the recall rate of the indicator fluorescent protein (F). (B) The workflow diagram shows the procedures of DCC-SMLM (Locs: localizations; COM: coefficient of mismatch; LCA: lateral chromatic aberration).